Isolating an unknown gene which encodes a desired peptide from a recombinant DNA library can be a difficult task. The use of hybridisation probes may facilitate the process, but their use is generally dependent on knowing at least a portion of the sequence of the gene which encodes the protein. When the sequence is not known, DNA libraries can be expressed in an expression vector, and antibodies have been used to screen plaques or colonies for the desired protein antigen. This procedure has been useful in screening small libraries, but rarely occurring sequences which are represented in less than about 1 in 105 clones, as is the case with rarely occurring cDNA molecules or synthetic peptides, can be easily missed, making screening libraries larger than 106 clones at best laborious and difficult. Screening larger libraries has required the development of methods designed to address the isolation of rarely occurring sequences, which are based on the co-selection of molecules, along with the DNAs that encode them. In vivo methods have been developed to screen large libraries, such as phage display and “peptides on plasmids” using lacI fusions of peptides.
Phage display is based on DNA libraries fused to the N-terminal end of filamentous bacteriophage coat proteins and their expression in a bacterial host resulting in the display of foreign peptides on the surface of the phage particle with the DNA encoding the fusion protein packaged in the phage particle (Smith G. P., 1985, Science 228: 1315-1317). Libraries of fusion proteins incorporated into phage, can then be selected for binding members against targets of interest (ligands). Bound phage can then be allowed to reinfect Escherichia coli (E. coli) bacteria and then amplified and the selection repeated, resulting in the enrichment of binding members (Parmley, S. F., & Smith, G. P. 1988., Gene 73: 305-318; Barrett R. W. et al., 1992, Analytical Biochemistry 204: 357-364 Williamson et al., Proc. Natl. Acad. Sci. USA, 90: 4141-4145; Marks et al., 1991, J. Mol. Biol. 222: 581-597).
LacI fusion plasmid display is based on the DNA binding ability of the lac repressor. Libraries of random peptides are fused to the C-terminal end of the lacI repressor protein. Linkage of the LacI-peptide fusion to its encoding DNA occurs via the lacO sequences on the plasmid, forming a stable peptide-LacI-peptide complex. These complexes are released from their host bacteria by cell lysis, and peptides of interest isolated by affinity purification on an immobilised receptor target. The plasmids thus isolated can then be reintroduced into E. coli by electroporation to amplify the selected population for additional rounds of screening (Cull, M. G. et al. 1992. Proc. Natl. Acad. Sci. U.S.A. 89:1865-1869).
These bacterial methods are limited by the size of the library that can be created by current methods of introducing DNA into host bacteria, the potential cellular toxicity of the expressed peptides introduced, and by the inability to introduce post-translational modifications, or to incorporate novel amino acids into the expressed peptide.
An entirely in vitro ribosome system has been described based on the linkage of peptides to the RNA encoding them through the ribosome (WO91/05058). Ribosome display has also been used for the selection of single-chain Fv antibody fragments (scFv) (Miatheakis, L. C. et al., 1994 Proc. Natl. Acad. Sci. USA, 91: 9022-9026; Hanes, J. & Pluckthun, A. 1997 Proc. Natl. Acad. Sci. USA, 94: 4937-4942). This method suffers from the lower stability of the RNA genetic material and the increased degradation likely under certain selection conditions where RNAse is likely to be present.
The in vitro method described by Griffiths and Tawvfik (WO 99/02671 and WO 00/40712) addresses some of these concerns by compartmentalizing DNA prior to expression of peptides, which then modify the DNA within the compartment. Peptides capable of modifications, resulting from enzymatic activity of interest, are then selected in a subsequent step. However, no direct selection of peptide binding activity is possible of both peptide and DNA without modification of the DNA encoding that peptide, and by releasing the modified DNA from the compartment.
Another prior art method, covalent display technology, or CDT, is described in WO9837186. This method relies on covalent linkage of protein to DNA to retain the linkage of genotype to phenotype, through the cis action of the crosslinking protein. This method teaches that two requirements are needed for successful use of this technique. Firstly, proteins are required which interact in vitro with the DNA sequence which encodes them (cis action), and secondly, said proteins must establish a covalent linkage to their own DNA template. This method suffers from the fact that the DNA is chemically modified which can prevent the recovery and identification of the binding peptide of interest.
There remains a need for more versatile in vitro methods of constructing peptide libraries in addition to the methods described above, which can allow direct selection of binding activity, as well as for enzymatic activity, and that allow efficient production of complex peptide structures, while still allowing recovery of intact genetic material encoding the peptide of interest.